This invention relates to mycoplasmas associated with the genito-urinary tract, believed to be the cause of nongonococcal urethritis, pelvic inflammatory diseases, septic abortion, and a wide array of diseases of other tissues. More specifically, embodiments of the present invention provide nucleic acid probes and compositions, methods for their use for the specific detection or identification of Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in clinical and other samples, and packaged components suitable for use as kits.
Mycoplasmas are small wall-less bacteria, primarily isolated from animal sources including humans. There are over 70 members of the genus Mycoplasma, and several related genera which are also characterized by small wall-less bacteria; these are Spiroplasma, Acholeplasma, Ureaplasma, Anaeroplasma, and Asteroleplasma. Only a handful of the species within these genera have been found associated with humans-some presumed to be xe2x80x9cnormal floraxe2x80x9d, others occasionally pathogenic, and still others always believed to be clinically significant. Among the mycoplasmas known to be pathogenic, Mycoplasma pneumoniae is historically the most well studied, it is the major infectious agent of primary atypical pneumonia. Nucleic acid compositions and methods for the detection of Mycoplasma pneumoniae and a further mycoplasma pathogen, Mycoplasma fermentans, are the subject of two concurrently filed applications U.S. Ser. No. 07/673,686 and U.S. Ser. No. 07/673,687, entitled xe2x80x9cNucleic Acid Probes For The Detection Of Mycoplasma Pneumoniaexe2x80x9d and xe2x80x9cNucleic Acid Probes For The Detection Of Mycoplasma Fermentans Or The Aids-Associated Virus-Like Infectious Agent.xe2x80x9d At least one inventor is common to all these applications and the present application.
Three other mycoplasma species, which can be isolated from the human genito-urinary tract, Mycoplasma hominis, Mycoplasma genitalium and Ureaplasma urealyticum, are somewhat more enigmatic in the clinical implications of the detection of these organisms in the human body. Ureaplasma urealyticum, for example, although it is implicated in significant and serious human morbidity and mortality, may be found in asymptomatic xe2x80x9cnormalxe2x80x9d individuals as well. The three species, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum will be referred to herein as the genital mycoplasmas.
Mycoplasma genitalium was initially isolated from the urethras of two males with nongonococcal urethritis (Tully, et. al., Int. J. Syst. Bacteriol. vol. 33, 1983). Subsequently, M. genitalium was isolated from the human respiratory tract, in co-culture with Mycoplasma pneumoniae (Baseman, et. al., J. Clinical Micro. vol.26, 1988). However, studies have shown (for example Hooton, et.al., Lancet, 1988) that M. genitalium plays a role in at least some of the cases of acute urethritis, particularly in homosexual males. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of Mycoplasma genitalium. Nucleic acid probes which recognize both M. genitalium and Mycoplasma pneumoniae, evolutionary close relatives, are described in an application filed concurrently.
Mycoplasma hominis has been implicated as a causative agent of salpingitis, amnionitis, nonspecific vaginitis, and postpartum septic fever. Mycoplasma hominis can be isolated from a significant number of asymptomatic women. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of Mycoplasma hominis. 
Ureaplasma urealyticum has been implicated in nongonococcal urethritis, chorioamnionitis, premature delivery, and perinatal morbidity and mortality. Some clinical investigators estimate that the etiological agency of nongonococcal urethritis (NGU) by Ureaplasma urealyticum may approach the same levels as that of Chlamydia trachomatis. As much as 40% of acute NGU may be caused by ureaplasma (Bowie, Urological Clinics of North America, vol. 11, 1984). This translates to approximately 3-4 million United States cases per year. Like Mycoplasma hominis, Ureaplasma urealyticum can be isolated from a significant number of both male and female asymptomatic individuals. There are at least 15 serotypes of Ureaplasma urealyticum. The combination of serotype, numerical prevalence and other factors that contribute to ureaplasma pathogenesis is, at present, totally unknown. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of Ureaplasma urealyticum. 
The mycoplasmas, such as those described above, are fastidious organisms, requiring complex culture media containing peptone, yeast extract, expensive animal sera, and sterol. Growth is relatively slow and reaches low cell densities compared to most bacteria. In addition, atmospheric conditions for cell growth require the addition of carbon dioxide. For these reasons, many clinical laboratories are unable to perform culture isolation of mycoplasmas, and consequently are left with no real ability to diagnose the presence of these important pathogenic bacteria. Given that mycoplasmas lack cell walls, antibiotics that target the bacterial cell wall, such as penicillin, have no anti-mycoplasma activity. Consequently, it is of importance for a physician to make a diagnosis of the presence of one or more of these bacteria, particularly if the clinical presentation is predictive, and prescribe the appropriate antibiotic.
Ribosomes are of profound importance to all organisms. Ribosomes serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacterium, including the mycoplasmas, and this convention will be continued herein. The probes of the present invention target the 16S and 23S rRNA molecules of the genital mycoplasmas.
Hybridization is a process by which, under appropriate reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together In an antiparallel fashion (one oriented 5xe2x80x2 to 3xe2x80x2, the other 3xe2x80x2 to 5xe2x80x2) to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under hybridization conditions, specifically and preferentially, to target nucleic acid sequences. The term xe2x80x9cpreferentiallyxe2x80x9d is used In a relative sense, one hybridization reaction product is more stable than another under identical conditions. Under some conditions, a hybridization reaction product may be formed with respect to one target, but not to another potential binding partner. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, biotin, etc.), to facilitate detection (e.g. chemiluminescent, fluorescent agents and radioactive agents) or to facilitate their capture onto a solid support (e.g., homopolymer xe2x80x9ctailsxe2x80x9d). Such modifications are elaborations on the basic probe function which is the probe""s ability to usefully discriminate between target and non-target organisms in a hybridization assay.
A minimum of ten nucleotides are necessary in order to statistically obtain specificity and form stable hybridization products. A maximum of 250 nucleotides represents an upper limit of sequences in which reaction parameters can be adjusted to determine mismatched sequences and preferential hybridization.
Hybridization conditions are defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Normal hybridization conditions for nucleic acid of 10 to 250 bases are a temperature of approximately 60xc2x0 C. in the presence of 1.08 M sodium chloride, 60 mM sodium phosphate, and 6 mM ethylenediamine tetraacetic acid (pH of 7.4).
Reaction parameters which are commonly adjusted include concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
Kohne et.al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences. However, Kohne et.al. do not provide the teaching necessary to make probes to detect the three species of genital mycoplasmas.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. Separately, or together, Kohne et al, Pace and Campbell, Sogin, Sogin and Woese, and Fox, Pechman and Woese fail to provide specific probes useful in assays for detecting the presence and abundance of genital mycoplasmas.
Hogan, et.al. (International Patent Application, Publication Number WO 88/03957) describe five probes for the specific detection of Mycoplasma pneumoniae. However, Hogan et.al. do not describe probes to the specific three genital mycoplasma species.
Woese, Maniloff, Zablen (Proc. Natl. Acad. Sci. USA vol. 77, 1980) examined the partial sequences of selected mycoplasma 16S rRNA, and define the concept of mycoplasma sequence xe2x80x9csignaturexe2x80x9d. However, Woese et.al. fail to teach the art of probe design. Woese et.al. do not discuss M. genitalium, M. hominis, or Ureaplasma urealyticum. 
Rogers, et.al. (Proc. Natl. Acad. Sci., USA, vol. 82, 1985) discuss sequences of 5S rRNA. However, Rogers et.al. fail to teach the art of probe design. Rogers et.al. do not mention M. genitalium or M. hominis. 
Weisburg, et.al. (Jnl. Bacteriology, vol.171, 1989) discuss 16S rRNA sequences of Ureaplasma urealyticum and M. hominis. However, Weisburg et.al. do not discuss or teach mycoplasma probe design.
Hyman, et al. (Hymen, et al., Jnl. Clin. Micro., vol. 25, 1987) and Jensen, et al. (Jensen, et al., Jnl. Clin. Micro. vol 29, 1991) discuss detection of Mycoplasma genitalium using polymerase chain reaction (PCR). Both papers fail to teach. non-PCR assays with respect to the utility of the sequences of mycoplasma rRNA or rDNA.
The present invention features nucleic acid compositions and composition sets, and methods for their use for the specific detection or identification of Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium. One embodiment of the present invention features, as a composition of matter, a nucleic acid having approximately 10 to 250 nucleotides capable of hybridizing to rRNA or rDNA of pathogenic mycoplasma bacteria associated with human urinary tract and genital areas, in preference to rRNA or rDNA of nonmycoplasma bacteria in humans. The nucleic acid composition is useful for detecting Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium. 
The nucleic acid composition are complementary to or homologous with a region of rRNA or rDNA selected from the group of regions consisting of positions 50 to 100, 425 to 485, or 1100 to 1150 of the Mycoplasma hominis 16S rRNA, positions 50 to 100, 150 to 250, 425 to 485, 800 to 850, 1090 to 1160, and 1220 to 1260 of the Ureaplasma urealyticum 16S rRNA, positions 1110 to 1160 of the Mycoplasma genitalium 16S rRNA, and positions 260 to 330, 1590 to 1630, and 1850 to 1900 of the Mycoplasma genitalium 23S rRNA. Ass such numerical designations are nucleotide positions counted from the 5xe2x80x2 end of the RNA molecule, a convention known to those skilled in the art.
Preferably, the nucleic acid composition is complementary to at least 90xc2x0 of a sequence comprising any ten consecutive nucleotides within a nucleic acid sequence selected from the group consisting of probes 2262 (Sequence No. 1), 2256 (Sequence No. 2), 2246 (Sequence No. 3), 2218 (Sequence No. 4), 2271 (Sequence No. 5), 2220 (Sequence No. 6), 2259 (Sequence No. 7), 2227 (Sequence No. 8), 2219 (Sequence No. 9), 2335 (Sequence No. 10), 2337 (Sequence No. 11), 2334 (Sequence No. 12), 2336 (Sequence No. 13), 2191 (Sequence No. 14), 2228 (Sequence No. 15), and 2260 (Sequence No. 16).
A further embodiment of the present invention includes a nucleic acid which is homologous to at least 90% of a sequence comprising any ten consecutive nucleotides within the sequences selected from the group consisting of probes 2262 (Sequence No. 1), 2256 (Sequence No. 2), 2246 (Sequence No. 3), 2218 (Sequence No. 4), 2271 (Sequence No. 5), 2220 (Sequence No. 6), 2259 (Sequence No. 7), 2227 (Sequence No. 8), 2219 (Sequence No. 9), 2335 (Sequence No. 10), 2337 (Sequence No. 11), 2334 (Sequence No. 12), 2336 (Sequence No. 13), 2191 (Sequence No. 14), 2228 (Sequence No. 15), and 2260 (Sequence No. 16).
One embodiment of the present invention features as an article of manufacture a set of at least two nucleic acid compositions. Each nucleic acid composition having approximately 10 to 250 nucleotides capable of hybridizing preferentially to rRNA or RDNA of Mycoplasma hominis, Mreaplasma urealyticum and Mycoplasma genitalium. Each nucleic acid is complementary to or homologous with at least 90%/ of a sequence comprising any ten consecutive nucleotides within the sequences selected from the group defined by probes consisting of 2262 (Sequence No. 1), 2256 (Sequence No. 2); 2246 (Sequence No. 3), 2218 (Sequence No. 4), 2271 (Sequence No. 5), 2220 (Sequence No. 6), 2259 (Sequence No. 7), 2227 (Sequence No. 8), 2219 (Sequence No. 9), 2335 (Sequence No. 10), 2337 (Sequence No. 11), 2334 Sequence No. 12), 2336 (Sequence No. 13), 2191 (Sequence No. 14), 2228 (Sequence No. 15), and 2260 (Sequence No. 16).
Probe sets suited for detecting Ureaplasma urealyticum includes probe 2262 (Sequence No. 1) and probe 2256 (Sequence No. 2); probe 2246 (Sequence No. 3) and probe 2218 (Sequence No. 4); probe 2259 (Sequence No. 7) and probe 2271 (Sequence No. 5); probe 2271 (Sequence No. 5) and probe 2220 (Sequence No. 6); and their complements.
Probe sets that are suited for detecting Mycoplasma hominis includes probe 2191 (Sequence No. 14) and probe 2228 (Sequence No. 15); probe 2228 (Sequence No. 15) and probe 2260 (Sequence No. 16); probe 2191 (Sequence No. 14) and probe 2260 (Sequence No. 16); and their complements.
Probe sets which are suitable for detecting Mycoplasma genitalium include probe sets 2227 (Sequence No. 8) and probe 2219 (Sequence No. 9); probe 2335 (Sequence No. 10) and probe 2337 (Sequence No. 11); probe 2335 (Sequence No. 10) and probe 2334 (Sequence No. 12); probe 2334 (Sequence No. 12); and probe 2336 (Sequence No. 13); and their complements. The probe 2227 (Sequence No. 8) and probe 2219 (Sequence No. 9) are capable of hybridization to the rRNA of the 16S ribosomal subunit. The probe sets probe 2335 (Sequence No. 10) and probe 2337 (Sequence No. 11); probe 2335 (Sequence No. 10) and probe 2334 (Sequence No. 12); probe 2334 (Sequence No. 12) and probe 2336 (Sequence No. 13) are capable of hybridizing to the 23S ribosomal subunit.
Embodiments of the present invention are also directed to methods for detecting Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium. The method includes contacting a sample potentially containing the microorganisms with at least one nucleic acid composition having 10 to 250 nucleotides capable of hybridizing preferentially to rRNA or rDNA of Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium. The base sequences, under conditions that allow the nucleic acid composition to hybridize, hybridize preferentially to rRNA or rDNA of the mycoplasma organism. Upon imposition of hybridization conditions on the sample, a hybridization product is formed in the presence of target. The detection of the hybridization product is an indication of the presence of the mycoplasma.
The method of the present invention features nucleic acid compositions which are complementary to or homologous with at least 90% of a 10 nucleotide sequence within the sequences selected from the group defined by probes consisting of 2219 (Sequence No. 9), 2262 (Sequence No. 1), 2256 (Sequence No. 2), 2246 (Sequence No. 3), 2218 (Sequence No. 4), 2271 (Sequence No. 5), 2220 (Sequence No. 6), 2259 (Sequence No. 7), 2227 (Sequence No. 8), 2335 (Sequence No. 10), 2337 (Sequence No. 11), 2334 (Sequence No. 12), 2336 (Sequence No. 13), 2191 (Sequence No. 14), 2228 (Sequence No. 15) and 2260 (Sequence No. 16).
Embodiments of the present invention also feature a method employing a first nucleic acid composition and a second nucleic acid composition. Each composition of different sequences, and each is complementary to or homologous with at least 90% of 10 nucleic acid sequences within the group of sequences defined by probes consisting of 2219 (Sequence No. 9), 2262 (Sequence No. 1), 2256 (Sequence No. 2), 2246 (Sequence No. 3), 2218 (Sequence No. 4), 2271 (Sequence No. 5), 2220 (Sequence No. 6), 2259 (Sequence No. 7), 2227 (Sequence No. 8), 2335 (Sequence No. 10), 2337 (Sequence No. 11), 2334 (Sequence No. 12), 2336 (Sequence No. 13), 2191 (Sequence No. 14), 2228 (Sequence No. 15) and 2260 (Sequence No. 16). Preferably the sequences of sets are probe 2262 (Sequence No. 1) and 2256 (Sequence No. 2); probe 2246 (Sequence No. 3) and probe 2218 (Sequence No. 4); probe 2259 (Sequence No. 7) and probe 2271 (Sequence No. 5); probe 2271 (Sequence No. 5) and probe 2220 (Sequence No. 6); probe 2191 (Sequence No. 14) and probe 2228 (Sequence No. 15); probe 2228 (Sequence No. 15) and probe 2260 (Sequence No. 16); probe 2191 (Sequence No. 14) and probe 2260 (Sequence No. 16); probe 2227 (Sequence No. 8) and probe 2219 (Sequence No. 9); probe 2335 (Sequence No. 10) and probe 2337 (Sequence No. 11); probe 2335 (Sequence No. 10) and probe 2334 (Sequence No. 12); and probe 2334 (Sequence No. 12) and probe 2336 (Sequence No. 13).
Individuals skilled in the art will recognize that the probe compositions of the present invention can be packaged with suitable instructions for their use as kits for detecting the presence of one or more of the organisms Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium. 
The nucleic acid compositions and the methods of the present invention provide the basis for nucleic acid hybridization assay for the specific detection of three of the presumptive etiological agents of nongonococcal urethritis, septic abortion, pelvic inflammatory disease, postpartum fever, in clinical samples such as genital swabs, genital lavage, sputum, throat swabs, blood, urine, cerebrospinal fluid, skin, biopsy, saliva, synovial fluid, bronchial wash, bronchial lavage, or other tissue or fluid samples from human patients or veterinary subjects. The nucleic acid compositions of the present invention also form the basis for confirmation of the presence of the genital mycoplasmas in liquid or semi-solid in vitro culture.
Embodiments of the present invention feature nucleic acid compositions capable of hybridizing to rRNA. Ribosomal RNAs constitute a significant component of cellular mass. Although estimates of cellular ribosome content vary, actively growing bacterial cells may contain upwards of 10,000 ribosomes per cell, and therefore 10,000 copies of each of the rRNAs present in a 1:1:1 stoichiometry in ribosomes. In contrast, other potential cellular target molecules such as genes or RNA transcripts thereof, are less ideal since they are present in much lower abundance. However, due to the close similarity of many organisms with which you may wish to distinguish mycoplasma species from, there can be no assurance that nucleic acid compositions can indeed be formulated which hybridize preferentially to the rRNA and RDNA of Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium. 
The discovery that probes could be generated with the extraordinary inclusivity and exclusivity characteristics of those of the present invention with respect to the detection of genital mycoplasma isolates, without necessarily incurring cross-reactivity between these species, or toward other bacterial taxa, except as documented, was unpredictable and unexpected.